Indian Journa l of Experimentai Biology Vol. 4 1, June 2003, pp. 574-580

Vasopressin mediates neuroprotection in mice by stimulation of V I vasopressin receptors: Influence of PI-3 kinase and gap junction inhibitors

Manoj G Tyagi* & K V Parthiban

Department of Pharmacology, Chri sti an Medica l Co llege, Vellore 632 002, India

Received 27 Decelllber 2002; revised 28 March 2003

europroteeti ve effect of vasopressin analogues, argin ine Vasopressin (A VP) and lysine Vasopressin (L VP) was evaluated against MgCl, induced cerebral ischemia model. A VP significantly prevented (P < 0.01 ) MgCl2 (1M) induced cerebral ischemia as compared to lysine Vasopressin (LVP) which was less effecti ve (P < 0.05 ). Pretreatment wi th PI -3 kinase inhibitors, Wortmannin and L Y -294002 (50 flg /kg, ip) significantl y altenu:lted the protecti ve effects of vasopress in . A VP was al so effective in reducing the maxima l electroshock (MES ) induced convulsive time and th is protective effect was blocked by PI-3 kinase inhi bitors. On the ot her hand, pretreatment with gap junction intracellular commu nication (GJl C) blocker, mepiIenamic acid (30 mg/kg, ip) significantly potentiated the MgCI, induced cerebra l ischemia. This enhancement of cerebra l isc hemia was not reversed hy vasopressin analogue, L VP. The role of V I vasopressin receptor was evaluated by pretreating the anima ls with non-selec ti ve VI receptor antago ni st, des Gly-NH2' d (CH2)5 [D-Tyr2, Thr 41 OVT whi ch re­versed the effects of A VP suggesting a role for vasopressin V I receptors. Thi s study suggests that neurohypophyseal hor­mone, A VP is nl:uroprotecti ve against MgClz induced cerebra l ischemia and thi s effect is modulated by PI-3 kinase en zyme

. inhibitors and protein kinase C inhibitors through possible influence on the cerebral vascular tone. This study suggests that ga p juncti ons have potential ro le in the induction of MgCl2 induced cerebral ischemia.

Keywords: Arg ini ne vasopress in, Cerebral ischemia, Gap junction, Lysine vasopress in , Neuroprotecti on, PJ-3 kinase, Protein kinase C, Vasopress in receptors

Cerebral ischem ia and haemorrhagic strokes resulting in ischemic ce ll death represent major cause of cere­brovasc ul ar di sorders. Various potenti a l treatment approaches have been developed to reduce the extent of tissue injury,approaches that have been derived from experimental models of cerebra l ischemia l.2 .

Neuronal damage mechanisms depend on complex interactions between neurotranmitters, neuropeptides and infl ammatory molecules that modify survival and repair. A pathololgical co nsequence of cerebral ischemia is the hyperactivity of spec ific neuronal syn­apses and mechanisms targeting atten uation of excito­toxicity at the molecular receptor, and/or ion channel, protein and enzy me changes conti nue to be explored for therapeutic implications3

. Drugs in tended for treatment of cerebral ischemi a might a lso be effective agai nst some for ms of epi lepsy. Some novel anticon­vulsant drugs emphasize this view as they also pre­vent cerebral ischemia due to constriction or occlu­sion of major cerebral arteri es4

-6

.

*Cnrrespondelll author: Tl:1 : (0416) - 262603 Fax : (0416) - 262788 E-ma il : lyagi243 @ yahoo.co.in

Arginine vasopress in (A VP) is an important hor­mone secreted by the posterior pitutary gland. A VP has also been shown to be a key regulator of physio­log ical homeostatic balance and acts on two types of vasopress in receptors V 1 and V2 in the brain. Vaso­pressin has been shown to have multifaceted action in the central nervous system affecting cogni­tion,behaviour and neuronal excitability7,8 . AVP has been fo und to induce depolarization of hippocampal neuro ns . A V P decreases the responsiveness of vaso­pressin neurons to acute changes in plasma osmo­lality. Data available o n A VP plasma level s in ischemic stroke are few and discordant5

. Several stud­ies suggest that A VP may have e ither pro- or anti­conv ul sant effect depending on the spec ific animal models of epilepsy , Vasopress in normally induces vasoco nstriction peripherally and also induces vaso­dilatation in some vascular beds by re leasing ni tric oxide and increasing cGMP levels9

.

On the other hand, PI-3 kinases induce the phos­phorylation of inositol contain ing lipids. PI, PI-4P and PI (4 ,5) P2 at the hydroxy l position on the inositol ring. A VP has been shown to acti vate the Pl-3 kinase enzyme in some cell types 10

-12 The enzyme have been

found to be expressed in the sy mpathetic and sensory

TYAGI & PARTHIBA N: VASOPRESSIN MEDIATES NEU ROPROTECTION IN MI CE 575

neurons also in discrete sites of the brain. Neurotro­phic factors for e.g. BDNF utili ses the PI-3 kinase pathways fo r eliciting excitation in hippocampal neu­rons. Wortmannin , a potent PI-3 kinase enzyme in­hibitor has been shown to inhibit long term potenita­tion in the dentate gyrus of the rats 13. Recent studies suggest that PI-3 kinase mi ght also contribute in the cytokine induced neuroendocrine responses in some cell types.

The importance of Mg2+ in homeostatic balance is well known. Mg2+ ion can cause inhibition of NMDA responscs in cul tured rat neurons and has been shown to pl ay all important role in neuronal depolarization. Mg2+ is an important ion for regul ati on of myogenic tone of cerebral blood vessels and it is be lieved to play an important role in the autoregulat ion of cere­bral blood flow. Dietary deficiency of magnesi um (Mg2+) as well as abnormalities in Mg2+ metabo li sm has been suggested to play importan t ro les in hyper­tensio n, stroke, atherosclerosis and diabeti c vascu lar disease I4

.15

.

We utilised MgCh for induci ng cerebral ischemia in tbis study. MgCl2 induced reversible global cerebral ischemia in mice is an excellen t ill vivo model l

(; .1 7

Recent studi es suggest an inten:ction of Mg2+ ion and PI-3 kinase enzyme in maintaining the cerebral vaSCLl-

, ~ . \

iar tone-). The purpose nf til!s study was to determ ine the ro le

of signa! Ii ng molecules in the neuroprotecti ve action of vasopressin an d to characterize the vasopressin receptor SUbtype responsible for neuroprorect ion. This study was hithcrto carried out to el ucidate the possible llcwoprotective role of vasopressin against MgCb induced cerebral ischemia and max imal elect roshock induced convuls ions in mice. The influe nce of PI-3 kinase enzyme activi ty, gap junc tions and nerve grow th fac tor was also evaluated.

Materials and Methods Drugs usedfor this study-- Norm al Sali ne (CMCH

Pharnncy , Vel lore), MgCi2 (G laxo India Ltd, Indi a), Argi ni ne Vasopressi n, Lys ine Vasopress in (S hree Ganesh Pharmaceuticals, India, Sigma India ltd), L Y 294002, Wonma nn in, Staurosporine and Nerve Growth Factor (A lol1lone Labs, Is rael), Mephenam ic Acid (Parke-Dav is Ltd. India). DesGly-NI-12 , d (Cl-1;1) S [D-Tyr2, Thr41 OVT (Dr.Maurice Man ni ng , Ohio, USA) .

Anilllal Corc- Thi s study was conducted on albino Swiss mice of either sex weighing between 25 and 3S g. The an imals were kept under standard

laboratory conditions and given food and water ad libitum . A 12 hr dark : li ght cycle was also main­tained.

Induclion of cerebral ischemia - Swiss albino mice were utili sed for inducti on of cerebral ischemi a. Global cerebral ischemia was induced by the in trave­nous inj ection (0.05 ml ) of 1M MgCb into the tail ve in 16.17 The indicati ve sy mptoms of cerebral ische­mia were gasping, sedation and loss of righting reflex . The total time duration of ischemic epi sode beginning with onset and recovery of ischemic episode W;'l:,

noted. Maximal electro shock method-- Max imal Electro

Shock seizures were induced in the animals usin g a I · d . b d I' 1 S 1'-) Th . I ~ tec lnlque escn e eard er ". e alllma s were pre-

treated with the tes t drugs pri or to the start of exper i­ments. The animals were subjected tu electro shock (60 mA/O.2 sec) via transauri cul ar electrodes attached to a electroconclusive meter. After induction of sei ­zures, durat ion of toni c con vu ls ion, clonic con vul sion and total time to regain righti ng reflex or mortali ty of the ani mals were noted and tabulated .

Biostatiscal evalualion- The data are represented <1:; mean ± SE. Statist ically signi ficant diffe rence vvas ascerta ined by ' ,0' va lue which is considered signifi­cUll t of P<O.OS and highl y sign ificant of P<O.Ol :IS

comparison of different groups were done uSing ANOV A and individual groups of Student's i test .

Results The results of our study conducted on cerebral

ischemia in the mice are shown in Table 1 and Figs1 to 4. The experiments were conducted on different groups of mice (n = 6). Our experiments suggest that

Table 1-- Errcci of intrape ritoneal injection or vasopressin an~l­

logues NG F and ~ taurospo rill e 011 MgCI 2 induced cerebra l ische­mia in mice . The animal, werc prctreated wi th A VP ail e! LVP ( 10 fig/kg ip) , NGF (60 ~lg/kg, ip) and st;tu rosporine (20 ~lg/kg , ip) 15 min before intravenous inject ion of MgC!1 (1M) .

ID:lta are me,l[l ± SE 1'01' groups of rats:

Prctrcatment (Dose, ip)

I) S,dine (0. 1 ml )

2) Sal ine (0. 1 ill l)

3) AVP

4 ) LVP

5) Sa lillc

6) AV P

Treatment (Duse, ip)

Sa li nc

NGF

NGF

NGF

St:ilIJ'Osprxi nc

Stau rosporinc

MgCl 2 induced cerebral ischemia

ti mc (sec)

100 (124.2 ± 3.43) 81.21 ± 2.73~:s

59.14 ± 3.1 i * 64 . 16 ± 4.07*

76.28 ± 3.63*

47.02 ± 1.97*"

*P<O.OS. **1'<0.01. NS p>O.OS. ------------------------

576 INDIAN J EXP SIOL, JUNE 2003

pre-treatment of AVP and LVP (10 I-lg/ kg, ip) pro­duced significant neuroprotection against 1M MgCh (0.05 ml, iv) induced ischemia i.e. a reduction in ischemic time by 78% and 37% respectively (P<O.OI, <0.05).

In order to characterize the sUbtype of vasopressin receptor involved in the action of vasopressin, we pre-

100

U 90

~ CIl 80 ~

E 70 u E 60 ~ .c

50 u <Jl

C 40 ~

'" to 30 ~ u ~ 20 "0

~ 10

0

AVP LVP V,A + LVP

Fig. 1 - Effect of pre-treatment with vasopress in anal ogues A YP, L YP (10 f1g/kg, ip) and non - selective vasopress in antagonist des Gly- NH2 (CH2) 5 D- [Tyr2, Thr4] OYT (100 f1g/ 100g, ip) on MgC I2 (1M, 0.05 ml , iv) induced cercbral ischemia in mice (n = 6). The data are represented as mean ± SE. ** P < 0.0 1 and *P < 0.05 .

100

(,) 9(} Q,) M gCI2 induchcl11i a In -IV

80

E - 70 u 'E 60 ~ .c u 50 '" C

40 Q)

Vl rll 30 Q) ... u 20 Q,)

"0

~ 0 10

0

Wortmannin + AVP L Y294002 +AVP

Fig. 2 - Influence of Wortmanni n and L Y 294 002 (50 f1g/kg, ip) on A YP (1 0 ~lg/kg, ip) induced neuroprotection against MgCI 2 ( 1M, 0.05 ml , iv) elicited cerebral ischemi a in mice (n = 6). The result s are expressed as mean ± SE.

treated the animals with a non- selective V t vaso­pressin receptor antagonist, DesGly-NH2' d (CH2) 5 [D-Tyr2, Thr4] OVT (100 I-lg/lOO g ip) and this an­tagonist reversed the protective actions of LVP. Thus signi fying the importance of V t vasopressin receptors. These results are displayed in Fig. 1.

To evaluate the role of PI-3 kinase enzymes we used two potent inhibitors of PI-3 kinase, wortmannin and L Y 294 002. Pretreatment of mice with PI-3 kinase inhibitors, Wortmannin and L Y294002 attenu­ated the neuroprotective actions of analogues A VP and LVP by 45 % and 54% respectively (P < O.Ol, < 0.05) . The results of these experiments are shown in Fig. 2.

200

180-u ., ~ 160-

Il> E 140-

u 120 E .,

.t::. 100-u .~ c 80-Il> If)

'" 60-e u .:: 40-

~ 20-

0-III

:.:.::~:.~::~~:.~

SALINE MEPHENAMIC LVP +

ACID MEPHENAMIC ACID

Fig. 3 - Effect of GJlC bl ocker, mephenam ic ac id (30 mg/kg, ip) on MgCl2 ( 1M, 0.05 ml , iv) induced cerebral ischemia in mi ce and in the presence of L YP (10 f1g/kg, ip). The results are expressed 3S

mean±SE. **P < O.Ol.

iii' 160 ",'0

c: c: o 0 140 .- " '" Q)

~~ 120 c: '" o E 100 ut= '0 c: " .- 80 u '" 60 " 0> ¥ ~ 40 <fl.J:: w U 20 :2: ~

0

Fig. 4 - Bar graphs illustrate effects of A YP (10 f1g/kg, ip) alone and in combinati on w i ~h L Y 294 002 and wortmann in (50 f1g/kg, ip) aga inst max imal electroshock induccd con vul sions in mice (n = 6). The da ta are depi cted as mean ± SE. * P < 0.05.

TYAGI & PARTHIBAN : VASOPRESSIN MEDIATES NEUROPROTECTION IN MICE 577

We utili sed mephenamic acid, a anthranilic acid analogue and GJIC inhibitor for evaluating our studies on gap junction intra cellular communication. Pre­treatment of mice with gap junction inhibitor, mephenamic acid (30 mg/kg, ip) potentiated the ac­tions of MgCl2 on cerebral ischemi a by increasing the ischemic time by almost 70% and thi s enhancement was significantly unaffected by L VP. The results are depicted in Fig. 3.

Table 1 illustrates the actions of NGF and Stauro­sporine against MgCl2 induced cerebral ischemia. A VP potenti ated the actions of NGF and the protein kinase C inhibitor, staurosporine against MgCl2 in­duced cerebral ischemia (P<0.05, < 0.01) .

A separate group of mi ce were pretreated with vasopressin and PI-3 kinase inhibitor to evaluate the effect of MES induced convulsions. A VP produced a 30.9% decrease (P < 0.05) in the convulsive time, while the PI-3 kinase inhibitors, Wortmannin and LY 294002 potentiated the MES induced convulsive time (P < 0.05 ) by 32% and 35% respectively. The results of these experiments are depicted in Fig. 4.

Discussion The results of our study demonstrate that vaso­

pressin analogues, A VP and LVP exhibit neuroprotec­tive action aga inst MgCl2 induced cerebral ischemia and maximal elctroshock induced convulsions in the mice. These results are shown in Fig.l.Vasopressin acts through the stimulation of specific V, and V2

vasopressin receptors expressed in the discrete sites of the brain and the peripheral ti ssues and blood vessels. Vasopressin contributes to blood pressure regulation in haemorrhagic conditions and might also contribute significantly in cerebral ischemic condition20. Intrave­nous injection of MgCl2 causes a state of reversible transient global ischemia characterized by gasping, sedation and loss of righting reflex . The animals re­cover from thi s state after few minutes. MgCh can induce cerebral ischemia by acting on NMDA recep­tors, affecting Ca2

+ entry or affecting activity of en­zymes e.g. PLA2 I PI-3 Kinase/PKC2,.22 . There is also possibly an increase in lipid peroxidation, which again is a consequence of stimulation by phospholi­pase enzymes like the PLA2 (s). Thus it is a safe and effective method of inducing ischemia.

On the other hand, pretreatment with vasopressin can prevent this ischemic episode by inducing vaso­constriction and increasing the blood pressure via the stimulation of V, receptors in the peripheral vascular beds and increas ing blood supply to the CNS. These

actions are dependent on the Ca2+ entry into the

vascular cells. Vasopressin also induces endothelium dependent vasodilatation in cerebral blood vessels by increasing the release of nitric oxide and cGMP levels23

.24 . Vasopressin receptors are expressed in the

brain stem region and particularly regul ate the responses of the Rostral ventro lateral medulla (RVLM). Stimulation of RVLM also protects the brain against ischemic insult by excitation of sympa­thetic system and elevating arterial pressure and inducing expiratory responses . Another important area in the brain stem termed the medullary cerebral vasodilator area or MCVA might also interact with RVLM for inducing a neuroprotective response by vasopressin. Both the vasopressin analogues A VP and LVP exhibit this neuroprotective act ion agai nst the MgCh induced cerebral ischemia. The results are de­picted in Fig. l.

Phospholipid levels of phosphatidylinositol biphosphate and monophosphate (PIP2) and (PIP) are reduced during 2-3 min of cei'ebral ischemia' . There­fore our next objective was to elucidate the role of PI-3 kinase enzymes because some recent studies suggest that these enzymes might contribute to seizure activity and also affect the tone of cerebral blood vessels25 . Vasopressin has been shown to stimulate the PI-3 kinase enzyme activity in several cell types26.27

. We utilised two potent inhibitors of the PI-3 kinase en­zy me. Wortmannin & L Y 294002 and pretreated the mice with these inhibitors along with the vasopressin analogues. Both these inhibitors are effective in studying the physiological role of the PI-3 kinase in in vivo models. The PI-3 kin ase enzyme inhibitors blocked the protective ac ti on of arginine vasopressin. PI-3 kinase enzymes are key signalling molecules in the neuronal cell survival pathway and are also in­volved in sympathetic neuronal activation28

. PI-3 kinases are also involved in the regulation of retro­grade axonal transport of nerve growth factor (NGF). Neurotrophic factors like Brain derived neurotrophic factor (BDNF) also utili se PI-3 kinase enzyme path­way. Thus it is suggested that these enzymes contri­bute effectively in maintaining the cerebral blood vessel tone during a state of cerebral ischemia. The results are shown in Fig. 4.

In order to characterize the vasopressin receptor subtype involved in the neuroprotecti ve action of vasopressin, we utili sed a nonselective V, receptor antagonist, des Gly NH2 d (CH2h[D- Tyr2,Thr4] OVT29. Pretreatment of mice with this V, receptor antagonist blocked the protective action of vasopressin

578 INDIAN J EXP BIOL, JUNE 2003

and caused an increase in ischemi c time. There was significant enhancement in the time of the loss of righting reflex . Thi s was indicative of the ro le for the V, vasopressin receptors. The results of this experi­ment are shown in Fig. 1.

Gap junctional communication can influence or­gani sm development, neuronal growth and homeo­stat ic control by sharing ions, second messengers and other signalling molecules3o

. Heterotrimeric gap junc­tion proteins are expressed in many types of vascular beds, we intended to investigate the role of intracellu ­lar gap junctional communication in modul ating MgCl2 induced cerebral ischemi a and for these ex­periments, we uti li sed mephenamic ac id (30mg/kg, i.p) belonging to the fenamate class of non steroidal anti-infl ammatory (NSAID) drugs3

' .

Experimental ev idence suggests that fenamates are important class of drugs wh ich are effective blockers of the Gap junction intracellular communication and they may block ani onic as well as cation ic channels. They also interact with connexins and indirectly per­turb the bulk membrane fluidity or the membrane pro­tein interface that wou ld affect the conformation of the connexins. The animals were pretreated with mephenamic ac id IS minutes before the injection of MgCI2, GJIC blocker mephenamic ac id caused an increase in the duration of ischemi c time and potenti­ated MgCb induced cerebral ischemia. This study illustrates that Gap junctions are critical for the ischemia inducing action of MgCb and blockade of GJIC by mephenamic acid causes potentiation and enhancement of the cerebral ischemi a. This is a novel finding suggesting the interaction of magnesium ion and gap junctional activity. This enhancement by mephenamic acid of the MgCl2 induced cerebral ischemia was not sign ificantly affected by pre­treatment with LVP.

NGF was the first di scovered and is the best known member of the neurotrophic factor family. NGF is produced in target tissues of the peripheral sympa­thetic and sensory nervous systems and binds to the tyrosine kinase receptor to elicit a cascade of intracel­lular events involved in neuronal differentiation, maturation and survival 32

. Ex pression of NGF corre­lates with the density of sympathetic innervation in effector organs and the amount of NGF can affect the sy mpathetic nerve survival and synaptic transmission between neurons and cardiac myocy tes. In this study NGF per se was unable to cause any significant change in the ischemia time, but pretreatment with A VP and LVP potentiated the effect of NGF, suggest-

ing an interaction of the vasopress in receptors with nerve growth factors. The results are depicted in Tab le 1

The concept that protein kinase C (PKC) plays a pivotal role in toni c contraction of smooth muscle is relatively recent. PKC is thought to phosphory late smooth muscle myosin and myos in light chain kinase in vitro and increase the Ca 2+ sensitiv ity of contractil e apparatu s and influence sustained phase of agoni st induced contraction. Earlier studies have shown the importance of PKC activation in cerebral arteri al con­trac tions. PKC act ivity is decreased after globa l ischemi a and have importance in cell necrosis '. Staurosporine has been shown to inhibit PKC activ­it/ o and block the long opening of L-type Ca2

+ chan­nels. Treatment with a PKC inhibitor staurosporine before an ischemic insult has protective effect. In this study , the non selective PKC inhibitor, staurosporine when injected (20 flg/kg , ip) in the mice potentiated the neuroprotecti ve effect of A VP (P < 0.05). These results are depicted in Tab le 1.

To examine the effect of vasopress in on the maxi­mal electroshock induced conv ul sions we utilised the swiss albino mice. The animals were treated with A VP and subjected to MES induced convulsions. A VP decreased the convulsive time significantly . Pretreatment with PI-3 kinase inhibitors produced an enhancement of the MES induced convulsive time (Fig. 4). These results demonstrate that A VP is pro­tective against MES induced cerebral convulsions. A VP has been shown to stimulate PI-3 kinase activity in various cell types. An important role for PI-3 kinase in neuronal excitability is well established. A maximal electroshock stimuli results in massive depo­larizat ion and PI-3 kinase inhibition possibly en­hances the post tetanic potential. Our experiments using these inhibitors of the PI-3 kinase suggest the possible role of PI- 3 kinase in modifyi ng the A VP response to MES conv ulsions. A VP reduces the inten­sity of MES convulsive disorder probably by mod ul ating the central opioidergic and alpha 2 adren-

. 33 34 erglc system . .

In conclusion, this study demonstrates that vaso­pressin analogues stimulate the vasopressin V, recep­tors and mediate neuroprotection against global cere­bral ischemia. In thi s process the signalling cascades involving the PI-3 kinase (s), and protein kinase C may significantly alter the effects of vasopressin. This study suggests that Gap junctions have a potential role in the induction of cerebral ischemia.

TYAGI & PARTHIBAN : VASOPRESS IN MEDIATES NEUROPROTECTION IN MICE 579

Acknowledgement The authors thank the Alomone Labs Ltd, Jerusa­

lem, Israel and the CMCH Pharmacy, Vellore for their generous gift of drugs and chemical s required for this study . The authors thank Dr. Maurice Man­ning, Med ical College of Ohio, Toledo, USA for the gift of non-selective vasopress in antagonist. The au­thors are grateful to Dr. JF Laycock, Neuroendocri­nology Department, Imperial College o f Medicine , London , UK for hi s useful suggesti ons regarding thi s study.

References I Lipton P, Ischcmi c cell death in brain neurons, Physiol Rev,

79 (1999) 143 1. 2 Bern aud in M, Mart i H H, Roussel S, Di voux D, Ncuvelot A,

MacKenzie E T & Petit E, A potential role for erythropoietin in foca l permanent cerebral ischemi a in mi cc, J Cereb Blood Flow Metab, 19 (1999) 643.

3 Hayashi T, Abc K & ltoyama K, Reduction of ischcmi c dam­agc by application of vascular cndotheli al growth factor in rat brain after transient ischemi a, J Cereb Blood Flow Metab , 18 (1998) 887 .

4 Smith S C, Mart in K F & Heal D J, Demonstration of neuro­protec tion by the novel anticonv ulsant BTS72664, after per­manent focal ccrebral ischemi a in rats, Br J Pharmacol (Pro­ceeding SlIpplJ, 124 ( 1998) 49.

5 Barreca T, Gandolfo C, Corsini G, Del-Sette M, Cataldi -A, Rolandi E & Franchin i R, Eva lution of the sec retory pattern of plasma argi nine vasopress in in stroke patients, Cereb Vasc Dis, II (200 1) 11 3.

6 Tyag i M G & Parthiban K V, Argin inc vasopressi n mediates neuroprotectio ll in the micc by st imulation of V I Vasopress in receptors; Influence of PI-3 kinase and gap junction inhi bi­tors, AIIII CO I((erellce of IABMS, Pondicherry, 0 I (2002) 47 .

7 Cheng S W T & North W G, Vasopressin red uces release from vasoprcssin neurons and Oxytocin neurons by ac ting on Vz like receptors, Brain Res, 479 (1989) 35 .

8 Tyagi M G & Thomas M, Enhanced cardi ovascul ar reactivity to des mopress in in water restri cted rats: faci litatory role of immunos uppress ion , Meth Find £xp Cl in Pharmacol, 21 (9) (1999) 6 19.

9 Garcia-Vi ll alon A L, Garci a J L, Fernandez N, Monge L, Gomez B & Dieguez G, Regional differences in the arterial response to vasopress in: role of endotheli al nitric ox ide, Br J Pham l[[col, 11 8 (1996) 1848.

10 Ramch L E & Cantley L C, The role of phosphoinositide 3 ki nase lipid products in cell function ,. J Bioi Chem, 274 (1999) 8347.

II Xu Y J, Ouk K S, Li ao D F, Katz S & Pelech S T, Stimula­tion of 90 and 70 kDa ribosomal protein 96 kinases by argin­ine vasopress in and ly sophosphatidic ac id in rat cardiomyo­cytes, Biochem Phannacol, 59 (2000) 11 63.

12 Ri ghi M, Tongiogi E & Cattaneo A, Brain derived neurotro­phic factor (BDNF) induces dendrit ic target ing of BDNF and tyros ine kinasc- B mRNAs in hippocampal neurones through a phosphatidyl inositol-3 kinase dependant pat hway, J Neuro Sci, 20 (2000) 3 164.

13 Kclly A & Lynch M A, Long term potentiati on in dentate gy rus of the ra t is inhi bited by Phosphoinosidate-3 kinase in­hibitor, Wortmannin , Neu ropharmacology, 39 (2000) 643.

14 Altu ra B M & Altura B T, Withdrawal of Magncsium in ­duces vasospasm wh il e elevated Magnesium produces re­laxat ion of tone in cerebral arteries, Neuro Sci Lell, 20 (1980) 323.

IS Yingy ing L S, Lev itan E S & John son J W, Free intrace ll ular Mg2+ concentration and inhi bition of NM DA responses in cultured rat neurons, J Physiol, 533 (200 1) 729.

16 Xing S H, Gao Y & Bian C F, Protcctive effccts of I-Tetra hyd ropalmitine on cerebral Ischemia in mice and Gerbil s, Asia Pacific J PharmClcol, 13 (1998) ISS.

17 Szabados T, Gigher G, Gecsalyi I, Gyertyan I & Levay G, Compari son of an ticon vul sivc and ac ute neruoprotec ti vc ac­ti vi ty of three 2,3-benzod iazepi nc compounds, GYK I 52466, GYKI 53405, and GYKI 53655, Brain Res 8 1,11, 55 (3) (200 1) 387 .

18 Tyagi MG & Jose VM. Anticonvulsant effects of cytoskele­tal depolymerizers in combin at ion with potassium opencr and adcnylate cyc lase activator: A causati ve link with nervc growth fac tor? Indiall J £xp Bioi, 39 (200 I) 425 .

19 Fi sher R S, Animal models of ep ilepsies, Brain Res Rev, 14 ( 1989) 245.

20 Laycock J F, Penn W P, Shirl ey D G & Walter S J, The role of vasopressin in blood pressure regulation immediatcly fo llow ing hemorrhage in the rat , J Physiol, 1979,296,267.

2 1 Daya nithi G, Widmer H & Ri chard P H, Vasopressin in­duced intracel lular Ca2+ increase in isolated rat supraopti c ce ll s, J Physiol, 490 ( 1996) 7 13.

22 Ruegg UT & GM Burgess. Staurospori ne, K 252, and UCN-0 1: Potcnt but non spec ifi c inhi bitors of protein kinases, Trends Pharll/acol Sci, 10 (1989) 2 I 8.

23 Oyama H, Suzuki Y, Satoh S, Kajita Y, Takayasu M, Shibu ya M & Sugita K, Role of nitric ox ide in the ccreb ra l vasod il utory responses to vasopressin and oxytocin in dogs, J Cereb Blood Flow Metab, 13 (1993) 285.

24 Katusic Z S, Shepherd J T & Van houtte P M, Vasopressin causes endothel ium dependent relaxation of the ca nine basi­lar arte ry, Circ Res, 55 (1984) 575

25 Yang Z W, Wang J, Zheng T, Altu ra B T & Altura B M, Low rM g2+]o induces contract ion and rCa2+]i ri ses in cerebral arteri es: Roles of Ca2+, PKC and PI -3 ki nase, Am J Physiol, 279 (2000) H2898.

26 Ni shi oka N, Akimoto K, Mori ya S, Takayanugi J, Fukui Y & Hirai S, Phosphat idyl inositol-3 kinase is in volved in TRE dependant gene response to arginine vasopressin , Biochelll Biophys Res.COI1lIllUI1, 2 15 (1995) 1037.

27 Ghosh P H, Mikhailova M, Beda lla R & Kreisberg J I, Ar­ginine vasopressi n stimulatcs mescngial cell proliferation by act ivat ing the epidermal growth factor receptor, Am J Physiol, 280 (200 I) F972.

28 Barl ett S E, Reynolds A J, Tan T, Heydon K & Hendry I A, Diffe rential mRNA ex pression and subcellular locati ons of PI-3 kinase isoforms in sy mpatheti c and sensory neurons, J Neurosci-Res, 56 (1999) 44 .

29 Mann ing M, Mitera K, Pancheva S, Stoev S, Wo N C & Chan W Y, Design and synthesis of highly selective ill vitro and ill vivo uterine rcceptor antagoni sts of oxytocin: comparison with Atosibun, 1111 J Peptide Protein Res, 46 (1995) 244.

30 Erik G A H, De Roos A D G, Peters P H J, Dehaan L H, Brower A, Ypey D L, Van Zoelen E J J & Theuvenet APR,

580 INDI AN J EXP BIOL, JUNE 2003

Fe namates: A novel class of reversible gap junction blockers, J Pharlll Exp Ther, 298 (200 I ) 1033.

31 He D S, Ji ang J X, Taffet S & Burt J M, Formation of hetero­tri meri c gap junction channels by connex ins 40 & 43 in vas­cular smooth muscle ce lls, Proc. Nat l Acad Sci USA, 96, (1999) 6495 .

32 Vai ll ant A R, Mazzoni I, Tudan C, Boudreau M, Kaplan D R & Miller F D, Depolariza ti on and ncurotransmi llers converge

on the phosphati dy linosito l 3 kinase Akt pathway to sy ner­gisti call y regulate neuronal survival, J Cell Bioi, 146 (1999) 955.

33 Vanti ni G, The pharmacologica l potenti al of neurot rophins : A perspecti ve, Psychol1eu roendocrino[ogy, 17 ( 1992) 40 I.

34 Ra maswamy S, Shewade D G, Tyagi M G & Bapna J S, Further studi es on the anti noc icept ive effects of vasopressin , J Pharlll Phan llacoi, 44 ( 1992) 698.